Mers-cov inhibitor peptides

ABSTRACT

The MERS-CoV inhibitor peptides include a set of peptides designed by modification or mutation of a wild type MERS-CoV fusion protein. The MERS-CoV inhibitor peptides are capable of inhibition of MERS-CoV membrane fusion, and thereby may prevent or slow the spread of MERS-CoV infections. Thus, the MERS-CoV inhibitor peptides may be used in pharmaceuticals to prevent and/or treat MERS-CoV infection. The pharmaceuticals may be formulated to comprise at least one of the MERS-CoV inhibitor peptides and a carrier, or they may include one or more expression systems capable of promoting cellular expression of one or more MERS-CoV inhibitor peptides. The MERS-CoV inhibitor peptides may also be used as reagents for MERS-CoV inhibition assays as a standard or reference inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 16/857,136, filedApr. 23, 2020, pending, the priority of which is claimed.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED IN COMPUTER READABLEFORM

The Applicants hereby incorporate by reference the sequence listingcontained in the ASCII text filed titled32087_14_Sequence_Listing_ST25.txt, created Mar. 13, 2020, and having 6KB of data.

BACKGROUND 1. Field

The disclosure of the present patent application relates to anti-RNAvirus peptides, and particularly to a MERS-CoV inhibitor peptides.

2. Description of the Related Art

Middle East Respiratory Syndrome Coronavirus (MERS-CoV), alternativelycalled HCoV-EMC/2012, causes severe respiratory illness with symptomsincluding fever, cough, and shortness of breath. Some subjects alsoexperience diarrhea, nausea, or vomiting. MERS-CoV is fatal for 3-4 ofevery ten people infected. Currently, there is no vaccine approved toprevent transmission of MERS-CoV and there is no specific antiviraltreatment suggested for MERS-CoV infection. Thus, preventative measuresgenerally involve routine avoidance of behaviors likely to lead toinfection (hand washing, covering the nose and mouth when sneezing,avoiding contact with the eyes, nose, or mouth, and avoiding directcontact with infected individuals). Care may include general medicalsupport for basic vital organ function, but does not include anymedications targeting MERS-CoV specifically.

MERS-CoV is an enveloped virus, which means a viral envelope proteinmust identify a host receptor and initial membrane fusion in order forthe virus to enter and infect host cells (membrane fusion may be eitherat the plasma membrane or in endosomes after endocytosis). MERS-CoVaccomplishes membrane fusion through interactions between thevirus'S-protein and host marker CD26.

Recent work in this field has focused on developing monoclonalantibodies to MERS-CoV, or screening pre-existing small moleculelibraries to look for compounds that inhibit S-protein mediated membranefusion. In addition, a peptide sequence found in the HR2 region of wildtype MERS-CoV has been shown to have some limited inhibitory effect onMERS-CoV membrane fusion. However, this prior work has yet to deliver apharmaceutical capable of either preventing MERS-CoV infection ortreating an infected subject.

Thus, MERS-CoV inhibitor peptides solving the aforementioned problemsare desired.

SUMMARY

The MERS-CoV inhibitor peptides include a set of peptides designed bymodification or mutation of a wild type MERS-CoV fusion protein. TheMERS-CoV inhibitor peptides are capable of inhibition of MERS-CoVinfection in cells and may be used to prevent and/or treat MERS-CoVinfection. The MERS-CoV inhibitor peptides may also be used as reagentsfor MERS-CoV inhibition assays as a standard or reference inhibitors.

An embodiment of the present subject matter is directed to apharmaceutical composition including one or more of the MERS-CoVinhibitor peptides and a pharmaceutically acceptable carrier.

An embodiment of the present subject matter is directed to a method ofmaking a pharmaceutical composition including mixing one or more of theMERS-CoV inhibitor peptides under sterile conditions with apharmaceutically acceptable carrier and preservatives, buffers, orpropellants to create the pharmaceutical composition; and providing thepharmaceutical composition in a form suitable for daily, weekly, ormonthly administration.

An embodiment of the present subject matter is directed to compositionsincluding one or more of the MERS-CoV inhibitor peptides and one or moreexpression systems. The expression system may be a viral basedexpression system, a plasmid based expression system, or any otherexpression system suitable for causing or enhancing expression of theMERS-CoV inhibitor peptides in a bacterium, yeast, or mammalian cell.The expression system may include a promoter sequence and DNA or RNAencoding one or more of the MERS-CoV inhibitor peptides.

An embodiment of the present subject matter is directed to methods ofinhibiting MERS-CoV infection, preventing MERS-CoV transmission, and/ortreating a MERS-CoV infection, including administering to a subject inneed thereof a therapeutically effective amount of a pharmaceuticalcomposition according to the present subject matter. In a furtherembodiment, the methods of inhibiting MERS-CoV infection may includepreventing MERS-CoV infection of a cell.

An embodiment of the present subject matter is directed to methods ofusing the MERS-CoV inhibitor peptides as reference agents to evaluateinhibition by other candidates against MERS CoV. These methods mayinclude using the MERS-CoV inhibitor peptides as reference agents inCell-Cell Fusion Assays, Viral Plaque Formation Assays, Viral RNAQuantitation Assays, or the like.

These and other features of the present subject matter will becomereadily apparent upon further review of the following specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a sequence alignment of Peptides 1-12 (SEQ ID NOs:1-12respectively) and of the consensus sequence (SEQ ID NO: 13) and displaysthe % conservation of each amino acid in the aligned sequences.

FIG. 2A depicts a graph of the results of a Cell-Cell Fusion Assaymeasuring the inhibitory properties of Peptides 1-12 at 0.1, 1, and 10μM.

FIG. 2B depicts a graph of the results of a Co-Transfection assayindicating that the Peptides have no interaction with the Cell-CellFusion Assay system.

FIGS. 3A-30 depict the results of a Plaque Reduction Assay for MERS-CoVtreated with 10 μM Peptides 1-12.

FIG. 4A depicts the results of a Plaque Reduction Assay for MERS-CoVtreated with Peptide 4 at 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM.

FIG. 4B depicts the results of a Plaque Reduction Assay for MERS-CoVtreated with Peptide 5 at 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM.

FIG. 4C depicts the results of a Plaque Reduction Assay for MERS-CoVtreated with Peptide 6 at 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM.

FIG. 5A depicts a graph of the effect of various concentrations ofPeptide 4 on the growth of Vero cells.

FIG. 5B depicts a graph of the effect of various concentrations ofPeptide 5 on the growth of Vero cells.

FIG. 5C depicts a graph of the effect of various concentrations ofPeptide 6 on the growth of Vero cells.

Similar reference characters denote corresponding features consistentlythroughout the attached drawings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The MERS-CoV inhibitor peptides include a set of peptides designed bymodification or mutation of a wild type MERS-CoV fusion protein. TheMERS-CoV inhibitor peptides are capable of inhibition of MERS-CoVinfection in cells and may be used to prevent and/or treat MERS-CoVinfection. The MERS-CoV inhibitor peptides may also be used as reagentsfor MERS-CoV inhibition assays as a standard or reference inhibitors.

Throughout this application, the term “about” may be used to indicatethat a value includes the standard deviation of error for thecomposition, device or method being employed to determine the value.

The use of the term “or” in the specification and claim(s) is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive, although the disclosuresupports a definition that refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps. In certain cases, the term “comprising” may bereplaced with “consisting essentially of” or “consisting of.”

The use of the word “a” or “an” when used herein in conjunction with theterm “comprising” in the claims and/or the specification may mean “one,”but it is also consistent with the meaning of “one or more,” “at leastone,” and “one or more than one.”

The phrase “pharmaceutically acceptable,” as used herein, refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a human.

The term “subject,” as used herein, means a mammal, including but notlimited to a human being.

As used herein, the term “providing” an agent is used to include“administering” the agent to a subject.

As used herein, a “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, isotonic and absorption delayingagents, buffers, carrier solutions, suspensions, colloids, excipients,and the like.

An embodiment of the present subject matter is directed to apharmaceutical composition comprising one or more of the MERS-CoVinhibitor peptides and a pharmaceutically acceptable carrier.

An embodiment of the present subject matter is directed to a method ofmaking a pharmaceutical composition including mixing one or more of theMERS-CoV inhibitor peptides with a pharmaceutically acceptable carrier.For example, the method of making a pharmaceutical composition caninclude mixing a MERS-CoV inhibitor peptide under sterile conditionswith a pharmaceutically acceptable carrier with preservatives, buffers,and/or propellants to create the pharmaceutical composition.

An embodiment of the present subject matter is directed to apharmaceutical composition including one or more of the MERS-CoVinhibitor peptides. To prepare the pharmaceutical composition, one ormore of the MERS-CoV inhibitor peptides, as the active ingredient, areintimately admixed with a pharmaceutically acceptable carrier accordingto conventional pharmaceutical compounding techniques. Carriers areinert pharmaceutical excipients, including, but not limited to, binders,suspending agents, lubricants, flavorings, sweeteners, preservatives,dyes, and coatings. In preparing compositions in oral dosage form, anyof the pharmaceutical carriers known in the art may be employed. Forexample, for liquid oral preparations, suitable carriers and additivesinclude water, glycols, oils, alcohols, flavoring agents, preservatives,coloring agents, and the like. Further, for solid oral preparations,suitable carriers and additives include starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents, and thelike.

The present compositions can be in unit dosage forms such as tablets,pills, capsules, powders, granules, ointments, sterile parenteralsolutions or suspensions, metered aerosol or liquid sprays, drops,ampules, auto-injector devices or suppositories, for oral parenteral,intranasal, sublingual or rectal administration, or for administrationby inhalation or insufflation. One or more of the MERS-CoV inhibitorpeptides can be mixed under sterile conditions with a pharmaceuticallyacceptable carrier and, if required, any needed preservatives, buffers,or propellants. The composition can be presented in a form suitable fordaily, weekly, or monthly administration. The pharmaceuticalcompositions herein will contain, per dosage unit, e.g., tablet,capsule, powder, injection, teaspoonful, suppository and the like, anamount of the active ingredient necessary to deliver an effective dose.A therapeutically effective amount of a MERS-CoV inhibitor peptide or anamount effective to treat a disease, such as a coronavirus infection,may be determined initially from the Examples described herein andadjusted for specific targeted diseases using routine methods.

An embodiment of the present subject matter is directed to compositionsincluding one or more of the MERS-CoV inhibitor peptides and one or moreexpression systems. The expression system may be a viral basedexpression system, a plasmid based expression system, or any otherexpression system suitable for causing or enhancing expression of theMERS-CoV inhibitor peptides in a bacterium, yeast, or mammalian cell.The expression system may include a promoter sequence and DNA or RNAencoding one or more of the MERS-CoV inhibitor peptides.

An embodiment of the present subject matter is directed to methods ofusing the MERS-CoV inhibitor peptides as reference agents to evaluateinhibition by other candidates against MERS CoV. These methods mayinclude using the MERS-CoV inhibitor peptides as reference agents inCell-Cell Fusion Assays, Viral Plaque Formation Assays, Viral RNAQuantitation Assays, or the like.

The MERS-CoV inhibitor peptides can be administered to a subject in needthereof. In an embodiment, the MERS-CoV inhibitor peptides can beadministered to a subject in need thereof to inhibit MERS-CoV infection,preventing MERS-CoV transmission, and/or treating a MERS-CoV infection.

An embodiment of the present subject matter is directed to a method ofinhibiting MERS-CoV infection, preventing MERS-CoV transmission, and/ortreating a MERS-CoV infection, comprising administering to a subject inneed thereof a therapeutically effective amount of the pharmaceuticalcomposition according to the present subject matter.

The MERS-CoV inhibitor peptides or pharmaceutical compositions thereofcan be administered to a subject by any suitable route. For example, thecompositions can be administered nasally, rectally, intracisternally,intraperitoneally, transdermally (as by powders, ointments, or drops),and/or parenterally. As used herein, “parenteral” administration refersto modes of administration other than through the gastrointestinaltract, which include intravenous, intramuscular, intraperitoneal,intrasternal, intramammary, intraocular, intrapulmonary, intrathecal,subcutaneous and intraarticular injection and infusion. Surgicalimplantation may also be contemplated, including, for example, embeddinga composition of the disclosure in the body such as, for example, in atissue, in the abdominal cavity, under the splenic capsule, brain, or inthe cornea.

Accordingly, the route of administration can include intranasaladministration, oral administration, inhalation administration,subcutaneous administration, transdermal administration, intradermaladministration, intra-arterial administration with or without occlusion,intracranial administration, intraventricular administration,intravenous administration, buccal administration, intraperitonealadministration, intraocular administration, intramuscularadministration, implantation administration, topical administration,intratumor administration, and/or central venous administration.

The MERS-CoV inhibitor peptides are designed by modification or mutationof a surface structure protein of MERS-CoV in the virus S2 spike region.The heptad repeat regions (HR1 and HR2) of S2 interact to help in fusionof MERS-CoV with cell membranes. The MERS-CoV inhibitor peptide S2 HR2derivatives were optimized to interfere with the proper mechanism ofHR1-HR2 interactions.

The following examples illustrate the present subject matter.

Example 1 Synthesis of MERS-CoV Inhibitor Peptides

The sequence of the HR2 region of wild type MERS-CoV is reported inTable 1 as Peptide 1 (SEQ ID NO: 1). This peptide was synthesized andused in assays as a reference or standard against which the activity ofnew peptides was compared.

To optimize a new sequence related to MERS-CoV HR2 with improved bindingpotency, computational studies were implemented to generate a set 684potential candidates by a series of mutations. Free energy-basedoptimization was computationally carried out to yield a set of topsuggested point mutations. Several point mutations were then generatedin different combinations with estimated improved binding strength andstability after testing by molecular dynamics experiments. Finally, aset of 11 peptides were synthesized (peptides 2-12 in Table 1).

Several systemic point mutations for every residue in the wild typepeptide were generated. Mutations were performed by replacing each aminoacid with any other member of the 21 known essential amino acids. Allmutations were generated only in MERS-CoV HR2. After each pointmutation, the free energy of binding of HR1 and mutated HR2 wascalculated as previously described. (Dehouck, Y. et al., “BeAtMuSiC:Prediction of changes in protein-protein binding affinity on mutations,”Nucleic Acids Research, 41: pp. W333-W339 (2013)). Candidates with thehighest values of binding free energy were synthesized for Anti-MERS-CoVtesting (FIG. 1 and Table 1). To confirm binding stability of eachpeptide after mutation, every HR1-HR2 complex was subjected to amolecular dynamics simulation as previously described. (Kandeel, M. etal., “Molecular dynamics of Middle East Respiratory Syndrome Coronavirus(MERS-CoV) fusion heptad repeat trimers,” Computational Biology andChemistry 75: pp. 205-212 (2018)).

A set of 12 peptides were obtained by custom synthesis service order toBiomatik Inc (Cambridge, ON, Canada) (Table 1). The peptides were HPLCpurified and confirmed by mass spectrum to ensure maximum purity andremoval of chemicals and byproducts during the peptide synthesisprocess.

TABLE 1The sequence of wild type and designed peptides (mutations in bold) NamePeptide Sequence SEQ ID NUMBER Peptide 1 (WT)SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLKEL SEQ ID NO: 1 Peptide 2SLTQINTTLLDLTYEMLSLQQVVKALNESYIDLK H L SEQ ID NO: 2 Peptide 3SLTQINTTLLDLTYEM K SLQQVVKALNESYIDLKEL SEQ ID NO: 3 Peptide 4 SLTQIN WTLLDLTYEM E SLQQVVKALNESYIDLKEL SEQ ID NO: 4 Peptide 5 SLTQIN WTLLDLTYEM E SLQQVVKALNE Y YIDLKEL SEQ ID NO: 5 Peptide 6 SLTQIN WILLDLTYEM E SLQQVVKALNE Y YIDLK H L SEQ ID NO: 6 Peptide 7 SLTQIN WTLLDLTYEM E SLQQV M KALNE Y YIDLK H L SEQ ID NO: 7 Peptide 8SLTQINTTLLDL E YEMLSLQQVVKALNESYIDLKEL SEQ ID NO: 8 Peptide 9SLTQINTTLLDL E YEM R SLQQVVKALNESYIDLKEL SEQ ID NO: 9 Peptide 10SLTQINTTLLDL E YEM R SL EE VVKALNESYIDLKEL SEQ ID NO: 10 Peptide 11SLTQINTTLLDL E YEM R SL EE VVK K LNESYIDLKEL SEQ ID NO: 11 Peptide 12SLTQINTTLLDL E YEM R SL EE VVK K LNESYID E KEL SEQ ID NO: 12

Example 2 Cell-Cell Fusion Assay of MERS-CoV Inhibitor Peptides

Cell-cell fusion assays were performed to quantitate the cell-cellfusion as described previously. (Yamamoto, M. et al., “Identification ofNafamostat as a Potent Inhibitor of Middle East Respiratory SyndromeCoronavirus S Protein-Mediated Membrane Fusion Using theSplit-Protein-Based Cell-Cell Fusion Assay,” Antimicrob. AgentsChemother. 60(11): pp. 6532-6539 (2016)). Briefly, a pair of 293FT-basedreporter cells, effector and target cells, that express individual splitreporters (DSP1-7 and DSP8-11 proteins) were used, because DSP1-7 andDSP8-11 produce fluorescence and luminescence only when the two proteinsform a tight complex. Effector cells stably expressing DSP8-11 andS-protein and target cells stably expressing DSP1-7 together with CD26and TMPRSS2 were prepared. Two hours before the fusion assay, botheffector and target cells were treated with 6 μM EnduRen (Promega,Madison, Wis., USA), a substrate for Renilla luciferase, to activateEnduRen. Each peptide was dissolved in 10% dimethyl sulfoxide (DMSO) andadded to 384-well plates (Greiner Bioscience, Frickenhausen, Germany)using a 12-stage workstation (Biotech, Tokyo, Japan). Next, a Multidropdispenser (Thermo Scientific, Waltham, Mass., USA) was used to add 50 μlof each single cell suspension (1.5×10⁴ effector and target cells) tothe wells. Incubation was performed at 37° C. for h, then RL activitymeasurements were obtained with a microplate reader (PHERAStar Plus, BMGLabtech, Cary, N.C., USA).

The results indicated strong inhibition of cell-cell fusion at 10 and 1μM (FIG. 2A). The most potent peptides were peptides 5, 8, 10 and 11,with EC50 values in low and middle nanomolar range (Table 2). Peptides5, 8, 10 and 11 showed stronger inhibition compared with peptide 1 (theoriginal wild type MERS-CoV sequence). A co-transfection control assaydemonstrated that the peptides did not interact with the assay system(See FIG. 2B).

TABLE 2 EC50 values of peptides by using cell-cell fusion assay PeptideEC50 (μM) 1 (SEQ. ID 1.055 NO: 1) 2 (SEQ. ID 0.916 NO: 2) 3 (SEQ. ID0.938 NO: 3) 4 (SEQ. ID 1.492 NO: 4) 5 (SEQ. ID 0.432 NO: 5) 6 (SEQ. ID0.917 NO: 6) 7 (SEQ. ID 2.355 NO: 7) 8(SEQ. ID 0.465 NO: 8) 9 (SEQ. ID1.299 NO: 9) 10 (SEQ. ID 0.426 NO: 10) 11 (SEQ. ID 0.039 NO: 11) 12(SEQ.ID 0.831 NO: 12)

Example 3 Plaque Assay of MERS-CoV Inhibitor Peptides

African green monkey kidney cells (Vero cells) were purchased from theAmerican Type Culture Collection (ATCC, Manassas, Va., USA). The cellculture was kept in a CO2 incubator at 37° C. in Dulbecco's modifiedEagle's medium (DMEM, Thermo Fisher Scientific, Waltham, Mass., USA)containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 25 mMHEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. MERS-CoV wasobtained from the Korea Centers for Disease Control and Prevention(CoV/KOR/KNIH/002_05_2015, Permission No. 1-001-MER-IS-2015001).

The plaque reduction assay was performed as reported previously. (Park,B. K. et al., “Generation and characterization of a monoclonal antibodyagainst MERS-CoV targeting the spike-protein using a synthetic peptideepitope-CpG-DNA-liposome complex,” BMB Rep. 52(6): pp. 397-402 (2019)).Briefly, Vero cells were cultivated on six-well plates for 12 h at 6×10⁵cells/well. In an initial study, MERS-CoV was mixed with each peptide ata final concentration of 10 μM for 30 min at 37° C. The mixtures ofMERS-CoV and each peptide were added to Vero cells in each well and thenincubated for 1 h. After incubation, the supernatants were removed andDMEM/F12 medium (Thermo Fisher Scientific) containing 0.6% oxoid agarwas transferred to each well. Four days after infection, plaqueformation was observed by staining with crystal violet and plaquenumbers were counted (See FIGS. 3A-30). The initial test resultsrevealed strong inhibition of MERS-CoV by peptides 4, 5, and 6. Theother peptides produced a more moderate decrease in plaque formation.Notable results include 98.3% inhibition of plaque formation by Peptide6, 98.2% inhibition by Peptide 4, 95% inhibition by Peptide 5, 74%inhibition by Peptide 2, and 69-70% inhibition by Peptides 11 and 12.(See Table 3)

TABLE 3 Plaque Inhibition Assay of Peptides 1-12 Percent of Sample #Plaques Normalize DMSO # Control 142 568 92.20779 DMSO 154 616 100Peptide 1 55 220 35.71429 (SEQ ID NO: 1) Peptide 2 36 144 23.37662 (SEQID NO: 2) Peptide 3 55 220 35.71429 (SEQ ID NO: 3) Peptide 4 11 111.785714 (SEQ ID NO: 4) Peptide 5 31 31 5.032468 (SEQ ID NO: 5) Peptide6 8 8 1.298701 (SEQ ID NO: 6) Peptide 7 55 220 35.71429 (SEQ ID NO: 7)Peptide 8 92 368 59.74026 (SEQ ID NO: 8) Peptide 9 81 324 52.5974 (SEQID NO: 9) Peptide 10 59 236 38.31169 (SEQ ID NO: 10) Peptide 11 46 18429.87013 (SEQ ID NO: 11) Peptide 12 48 192 31.16883 (SEQ ID NO: 12)

After initial confirmation of the inhibitory properties of the peptides,Peptides 4, 5, and 6 were tested in varying concentrations, ranging from50 μM to 3.125 μM (See FIGS. 4A-4C). The results of these experimentswere then used to calculate the half maximal effective concentration forPeptides 4, 5, and 6. The results are provided in Table 4.

TABLE 4 EC50 of Peptides 4, 5, and 6. Sample EC50 (μM) Peptide 4 0.302Peptide 5 1.428 Peptide 6 1.849

Example 4 Cytotoxicity and Viability

Vero cells (1×10³ per well) were plated on 96-well plates and culturedfor 12 h. The cells were treated with three fold serial dilutions ofPeptides 4, 5, or 6, or with 10% DMSO (control) for 3 days (Peptideconcentrations ranged from 100 μM to 0.4 μM). Then, cells were treatedwith 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,Sigma-Aldrich, St. Louis, Mo., USA) for 4 h at 37° C. Formazan crystalswere dissolved in DMSO, and the absorbance at 570 nm was measured usinga microplate reader (Thermo Fisher Scientific, Ratastie, Finland).

No cytotoxicity at concentrations equal to or lower than 10 μM wasobserved for any of the tested peptides (See FIGS. 5A-5C). Thus, it wasconcluded that Peptides 4, 5 and 6 have a safe cellular profile.

It is to be understood that the MERS-CoV Inhibitor Peptides are notlimited to the specific embodiments described above, but encompasses anyand all embodiments within the scope of the generic language of thefollowing claims enabled by the embodiments described herein, orotherwise shown in the drawings or described above in terms sufficientto enable one of ordinary skill in the art to make and use the claimedsubject matter.

1-5. (canceled)
 6. A method for conducting a MERS CoV inhibition assaycomprising using a MERS CoV inhibitor as a standard or as a referenceinhibitor, wherein the MERS CoV inhibitor comprises a peptide having theamino acid sequence selected from the group consisting of SEQ ID NOs:4-6 and a combination thereof.
 7. The method of claim 6, wherein theassay is a cell-cell fusion assay.
 8. The method of claim 6, wherein thethe assays is a MERS CoV plaque formation assay.
 9. A pharmaceuticalcomposition consisting of a MERS CoV inhibitor and a pharmaceuticallyacceptable carrier, wherein the MERS CoV inhibitor comprises a peptidehaving the amino acid sequence selected from the group consisting of SEQID NOs: 4-6 and a combination thereof. 10-14. (canceled)
 15. A method ofpreventing MERS CoV infection of a cell comprising: administering acomposition comprising at least one peptide having an amino acidsequence selected from the group consisting of SEQ ID NOs: 4-6, and acombination thereof. 16.-19. (canceled)